Field of the Invention
Embodiments relate to diagnostic testing for coeliac disease, sprue, and/or gluten sensitivity.
Background of the Related Art
Coeliac disease, also known as gluten-sensitive enteropathy or sprue, is a chronic inflammation of the small intestine often associated with extensive destruction of the intestinal epithelial cells, which may result in a malabsorption syndrome and/or associated immunological disorders.
The inflammation, if the corresponding genetic predisposition is present, is activated by a hypersensitivity to gluten, a collective term for the “sticky proteins” present in many cereal types. In wheat, the immunogenic prolamin (the alcohol-soluble fraction) is called gliadin. Homologous prolamins are also known in rye (secalins), barley (hordeins) and oats (avenins). In persons with the appropriate predisposition, these peptide fragments can result in a complex reaction of the intestinal mucosa and of the immune system.
The term gluten also covers gluten derivatives, gliadin or gliadin homologue extracts, non-synthetically or synthetically produced gliadin peptides and chemical derivatives of these known as “gliadin peptide”.
Certain gliadin peptides or homologous prolamin peptide fragments are absorbed by antigen-presenting cells, processed and loaded onto HLA molecules or bind directly to HLA molecules, principally to HLA-DQ2 or HLA-DQ8. This binding is reinforced by the deamidation of the amino acid glutamine that is frequently present in the peptide. This deamidation is principally catalysed via the enzyme tissue transglutaminase (tTg), and in particular by tissue transglutaminase 2 (tTg2). In addition the enzyme also catalyses a transamidation, by means of which a cross-linking of the surrounding peptides and proteins is induced. The deamidation of gliadin peptides guarantees high-affinity binding between the gliadin peptide and, for example, HLA-DQ2.
This peptide-HLA complex in turn binds to the CD4+T helper cells, which in turn leads to activation of the inflammatory cytokine cascade. This increases the production of various inflammation-triggering messenger substances, such as interferon-gamma, TNF-alpha, interleukin-6 and interleukin-2.
In the course of the inflammation, various antibodies are formed which target, for example, gliadin itself (gliadin antibodies) or tissue transglutaminase (tTg antibodies). Ultimately the inflammation process ends in the apoptosis of the enterocytes (lining cells of the epithelium of the small intestine), which leads to a more or less pronounced loss of small intestinal villi (villous atrophy). This results in the mucosa of the small intestine no longer being in a position to transfer sufficient nutrients from the intestine to the bloodstream, resulting ultimately in a malabsorption syndrome.
The prevalence (number of sufferers at the time of investigation/number of “studied” individuals) of coeliac disease or endemic sprue is 1:100, more recent studies, however, assume a prevalence of approx. 1:50. All in all, coeliac disease or endemic sprue reaches a frequency of 1% to 3% within the Caucasian population group.
Coeliac disease exhibits two manifestation peaks, one in infancy from approx. 6 to 18 months, one to three months after cereals have been included in the diet and a second peak aged approx. 30 to 40 years.
The most common symptoms of coeliac disease or sprue are chronic diarrhoea, iron deficiency with or without anaemia and osteopenia, cause by the digestive disorder. Failure to thrive can often be observed in affected children. Less common symptoms include chronic fatigue, nervousness, osteoporosis, osteomalacia, anxiety disorders, depression, muscle cramps and secondary lactose intolerance. Furthermore, various concomitant diseases may occur, such as selective IgA deficiency, dermatitis herpetiformis, diabetes Type I and gastrointestinal malignoma.
Diagnosis of coeliac disease or sprue is preferably or usually performed by means of a serological diagnosis and/or by means of histology of biopsies of the small intestine. The serological diagnosis of coeliac disease or sprue is normally tested for the presence of antibodies against a foreign antigen, such as gliadin (anti-gliadin antibodies) or deamidated gliadin peptide (anti-DGP antibodies) and against autoantigens of the endomysium (anti-EMA antibodies), but especially against tissue transglutaminase 2 (anti-tTg2 antibodies) or a complex of tissue transglutaminase and gliadin (anti-tTg/gliadin peptide complex antibodies).
Auto antibodies against endomysial antigens are highly specific and can be demonstrated in over 90% of patients with coeliac disease or with endemic sprue. In this case, an indirect immunofluorescence test on tissue sections from primate oesophagus is used. The anti-endomysium concentrations reflect the histological appearance: the higher the antibody titres are, the more pronounced is the villous atrophy. However, anti-endomysium detection by means of the immunofluorescence technique is fairly costly, as this test requires the user to have a high degree of technical competence, is time consuming and requires relatively rare biological material (primate oesophagus tissue).
From a historical perspective, the detection of antibodies against gliadin offered the first opportunity of identifying coeliac disease or endemic sprue by means of an antibody test procedure. However, anti-gliadin antibodies are not specific enough to detect endemic sprue to a satisfactory extent. The detection sensitivity was improved thanks to the discovery of anti-deamidated gliadin peptide (DGP) antibodies. A further improvement in the diagnosis and clinical monitoring of this disorder was the discovery of anti-tissue transglutaminase antibodies.
When it comes to the serological diagnosis of coeliac disease, the presence of anti-endomysial antibodies is usually detected.
A central aspect of coeliac disease diagnosis was, and to some extent remains, a histological examination of a biopsy of the small intestine, whereby the histopathological spectrum may range from minimum elevation of intraepithelial lymphocytes to complete villous atrophy. The characteristic histopathological elements for the above are: an increase in intraepithelial lymphocytes as well an increase in lymphocytes and plasma cells in the lamina propria, frequently mixed with eosinophils; reduced villi length, deepening of the intestinal crypts; reduced villi: Crypt ratio (normally >4-5:1); increased mitotic cell count; abnormal enterocytes (cuboid rather than cylindrical cells, loss of basal core polarity, loss of the brush border).
The histological grading is usually performed on the basis of the classification proposed by Marsh.
TABLEDistribution of the Marsh criteria [A. Vècsei et al. 2011, p. 6]TypeCryptsVilli0normalnormal1normalnormal2hyperplasticnormal3ahyperplasticslightly truncated3bhyperplasticseverely truncated3chyperplastictotally absent
On the basis of the diagnosis of symptoms, histological findings and serological findings, as well as genetic and clinical information, coeliac disease or endemic sprue can be assigned to various clinical sub-groups. These range from what is known as “silent sprue”, to “latent sprue”, and right through to manifest or classic sprue.
An early diagnosis of coeliac disease or endemic sprue is crucial to the progression of the disease. Following reliable and early diagnosis, the disease can be kept in remission through consistent observance of a gluten-free diet and the risks of concomitant and/or secondary illnesses, such as increased malignoma risk, can be prevented.
Therefore continuing to improve diagnostic tests for coeliac disease or endemic sprue is of primary concern. When it comes to a rapid, easy-to-perform, economical test, serological diagnostic procedures appear to be particularly suitable.
For example, EP 0 912 898 B1 teaches an immunological diagnostic procedure for antibodies that target tissue transglutaminase (tTg). In this diagnostic procedure, antibodies against tissue transglutaminase (tTG) from body fluids are demonstrated by means of an immune reaction with tissue transglutaminase, whereby the immune reaction is not performed with a tissue section from animal or human tissue.
Furthermore, test procedures are available which can detect antibodies which target a complex of tissue transglutaminase (tTG) and gliadin peptides. Such test procedures are, for example, offered by the company AESKU.Diagnostics. It has been shown with this test procedure that antibody formation can be proved before all other state-of-the-art tests.
Furthermore, even gluten intolerance or sensitivity is identified, which is not induced by coeliac disease. A distinction should be made between this disease, which is neither an autoimmune disorder or a wheat allergy, and coeliac disease. Both must first be excluded before it can be diagnosed. Furthermore, it must be determined as to whether there is any improvement with a gluten-free diet. No biomarkers have previously been identified for this disorder.